Normalisation of microarray data based on hybridisation with an internal reference

ABSTRACT

The invention relates to methods and corresponding arrays especially suited to correct for signal errors due to variations in sample preparation. Methods and compositions for performing quantitative array-based assays are provided. In the subject methods, both a reporter and an analyte is employed, where the reporter is characterized by binding selectively to an internal reference present on the array, i.e. at least a subset of, if not all of, the spots present on the array employed in the method contain an internal reference which can be bound by reporter.

FIELD OF THE INVENTION

The invention relates to methods and corresponding arrays especiallysuited to correct for signal errors due to variations in samplepreparation. Methods and compositions for performing quantitativearray-based assays are provided. In the subject methods, both a reporterand an analyte is employed, where the reporter is characterized bybinding selectively to an internal reference present on the array, i.e.at least a subset of, if not all of, the spots present on the arrayemployed in the method contain an internal reference which can be boundby the reporter.

BACKGROUND OF THE INVENTION

Microarrays of binding agents, such as oligonucleotides and peptides,have become an increasingly important tool in the biotechnology industryand related fields. These binding agent arrays, in which a plurality ofbinding agents are deposited onto a substrate, often a solid substrate,in the form of an array or pattern, find use in a variety ofapplications, including drug screening, nucleic acid sequencing,mutation analysis, genotyping, expression profiling, genetic abnormalityscreening by MAPH and the like. One important use of microarrays is inthe analysis of differential gene expression, where the expression ofgenes in different cells, normally a cell of interest and a control, iscompared and any discrepancies in expression are identified. In suchassays, the presence of discrepancies indicates a difference in theclasses of genes expressed in the cells being compared.

In methods of differential gene expression, arrays find use by servingas a substrate to which “probe” fragments or “receptors”, such as forexample polynucleotides, are bound. One then obtains “targets” or“analytes” from analogous cells, tissues or organs of, e.g. a healthyand diseased organism. The targets are next hybridized to theimmobilized set of polynucleotide “probe” fragments. Differences betweenthe resultant hybridization patterns are subsequently detected andrelated to differences in gene expression in the two sources.

Because of the varied and important information that microarrays canprovide, as well as the many potential applications of such devices, theuse of these microarrays in research, diagnostic and relatedapplications has grown considerably and is expected to continue to doso. A variety of different array technologies have been developed inorder to meet the growing need of the biotechnology industry, asevidenced by the extensive number of patents and other literaturepublished.

However, there are disadvantages with current protocols. For example,the efficiency of hybridization of target nucleic acids to the array canbe limited by experimental limitations, e.g. differences in samplepreparation or different target nucleic acids can have differenthybridization efficiencies to the probe nucleic acids of the array.Differences in hybridization efficiency result in differences in theintensity of hybridization to different probe nucleic acids of thearray, even though the targets are present in equivalent concentrations.Where two or more arrays are employed in a particular application, e.g.in gene expression analysis, variation in the quality of array(reproducibility of array production), and in assay conditions betweenthe different arrays can preclude direct comparison of data obtained onthe arrays, since conditions such as hybridization time, probe labeling,and detection procedures may differ, and variations between the arraysmay be present. All of these errors result in spot to spot variation.Furthermore, it is difficult to compare data generated by usingdifferent types of oligonucleotide or polynucleotide based arrays.Concentration of target nucleic acids in a sample cannot be comparedbetween arrays produced by different methods and/or manufacturers basedon intensity of signals because the set of probe sequences often differsbetween arrays. Thus, the signal error obtained in arrays is the sum ofall the individual errors such as the inhomogeneous substrateactivation, liquid dispense volume variation, probe couplingdifferences, temperature variation, flow variation, optical aberrations,et cetera. As a result, current array technology is used mainly fordiscovery of differentially-expressed genes rather than for any specificquantitative assay. In this respect, two formats are generally employed:(a) comparison of two hybridization patterns to each other and (b)simultaneous hybridization to the same array of two different targetsderived from two different biological sources and labeled by differentlabels. In the latter approach, which is more commonly employed, folddifferences in gene expression between the two samples are oftenmeasured.

In these application areas, as well as others, it is important tosignificantly distinguish between different mRNA expression levels andgenetic copy number differences as small as 1.5 fold. This requires thatall aspects of the system should be discriminative over a signaldifference of only 1.5 fold. As such, there is a continued need for thedevelopment of additional arrays and array-based protocols.

Of interest would be the development of an array-based methodology thatincorporates an internal calibration standard, where such a method wouldeliminate variations resulting from the quality of the array, the typeof the array, the quality of the assay conditions, and the like. Inaddition, there is a need for an array-based protocol that providesquantitative data about sample preparation, target concentration, and acorresponding method of quantification to allow more accurate comparisonof data between arrays.

WO 00/34523 by Hyseq Inc describes the addition of a detectable labelwhich is proportional to the amount immobilized at a certain spot, tocorrect for probe coupling differences during the preparation of theassays. Similarly, WO 00/65095 by Clontech Laboratories Inc relates tothe normalisation for differences in immobilization efficiencies of theprobes at different addresses in the array.

Evidently, the prior art relates only to the manufacture of arrays andspot to spot variation, but not to signal errors due to analyteprocessing. Meanwhile, contemporary microarrays are produced byestablished techniques, resulting generally in qualitatively highlyreliable products. The predominant quality problem resides in the samplepreparation.

SUMMARY OF THE INVENTION

In order to measure individual differences and subsequently correct forthese differences the present invention provides microarrays comprisinga substrate with predefined regions, wherein each binding substanceimmobilized at a predefined region of said substrate comprises areceptor and a predetermined amount of an internal reference, whereinthe signal generated by said internal reference is determined by meansof a reporter molecule and wherein said reporter molecule selectivelybinds to said internal reference. Moreover, the present inventionprovides a method for the identification of an analyte in a sample, suchas for example a biological sample, comprising the steps of:

-   -   (a) providing a microarray comprising a substrate wherein each        binding substance immobilized onto said substrate comprises a        predetermined amount of receptor and a predetermined amount of        an internal reference,    -   (b) providing a reporter molecule that binds selectively to said        internal reference,    -   (c) adding said reporter molecule to said sample,    -   (d) contacting said sample comprising said reporter molecule        with said microarray under conditions that allow binding to take        place between said receptor and said analyte, and between said        internal reference and said reporter molecule,    -   (e) determining the signal of said reporter molecule binding to        said internal reference,    -   (f) determining the signal of said analyte binding to said        receptor, and,    -   (g) normalising said signal of step (f) for said signal of step        (e).

DETAILED DESCRIPTION

The invention relates to methods and corresponding microarraysespecially suited to correct for signal errors due to variations insample preparation. Methods and compositions for performing quantitativemicroarray-based assays are provided. In the subject methods, both areporter and an analyte is employed, where the reporter is characterizedby binding selectively to an internal reference present on the array,i.e. at least a subset of, if not all of, the spots present on the arrayemployed in the method contain an internal reference which can be boundby the reporter.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural references unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Generally, in order to analyse a sample on a microarray, the sample ismanipulated before it is contacted to said microarray. Manipulationsinclude, for example, cDNA production from RNA, production and/orisolation of nucleic acids, antibodies, polypeptides and the like. Eachstep of this manipulation of a sample can introduce errors, such as inamount or integrity of the molecule of interest. This problem isextremely manifest if two or more samples need to be compared. Thepresent invention relates to the normalisation of signals of samplescontaining an analyte via adding a predetermined amount of a reporter tothis sample. The internal reference is eventually used for normalisingsample to sample variation due to the processing of the samples (betweensamples normalisation), as well as variations observed with one subjectsample, such as for example due to spot to spot variation in a givenmicroarray (within sample normalisation).

In particular, the present invention provides a method for theidentification of an analyte in a sample comprising the steps of:

-   -   (a) providing a microarray comprising a substrate wherein each        binding substance immobilized onto said substrate comprises a        predetermined amount of receptor and a predetermined amount of        an internal reference,    -   (b) providing a reporter molecule that binds selectively to said        internal reference,    -   (c) adding said reporter molecule to said sample,    -   (d) contacting said sample comprising said reporter molecule        with said microarray under conditions that allow binding to take        place between said receptor and said analyte, and between said        internal reference and said reporter molecule,    -   (e) determining the signal of said reporter molecule binding to        said internal reference,    -   (f) determining the signal of said analyte binding to said        receptor, and,    -   (g) normalising said signal of step (f) for said signal of step        (e).

The term “analyte in a sample” refers to a molecule in a sample, i.e. amolecule to be analysed which is present in a sample. The molecules in asample can be, e.g. nucleic acids (both DNA and RNA), peptides,polypeptides, proteins, antibodies, carbohydrates, and or smallbiomolecules (e.g. drug candidates). The sample can be, for example, aphysiological or a biological sample.

Samples are generally manipulated in order to isolate and/orcharacterise the analyte. For example, analyte nucleic acids aregenerally isolated from a biological sample (cells, tissues, organs,etc.), processed and converted to other nucleic acids using known in theart technology, such as PCR, reverse transcription, etc., e.g. mRNA,cDNA, PCR products, cRNA, and the like. The analyte nucleic acids may beisolated from a tissue or cell of interest using any method known in theart. Total RNA or its transcriptionally active fraction mRNA can beisolated from a tissue and labeled and used directly as analyte nucleicacid, or it may be converted to a labeled cDNA, cRNA, etc. via methodssuch as reverse transcription, transcription, Tyras, NASBA and/or PCR.Generally, such methods will employ the use of oligonucleotide primers,and the primers can be anchored by bacteriophage RNA polymerasepromoter. The primers may be designed to copy a large spectrum of RNAspecies, e.g. oligo (dT) primers or random hexamers, or designedspecifically to copy a subset of genes of interest. After the copyingstep, i.e. conversion of mRNA to cDNA, cDNA can be amplified by PCR orby linear amplification using bacteriophage RNA polymerase mediatedtranscription, NASBA or Tyras. As with the reporter nucleic acids, in apreferred embodiment the analyte nucleic acid sequences are generatedusing a set of a representative number of gene specific primers.

In the present invention, the term “reporter” refers to a molecule thatcorresponds, e.g. interacts with or binds to, an internal reference,which is covalently bound to the substrate of the array. The reporter isadded to a sample, before said sample is contacted to an array. Thereporter can be added at different steps of sample manipulation. It willbe clear that if a reporter is added in the first step(s) of samplemanipulation, then the reporter will undergo the same or most of thesteps of the manipulation which the sample undergoes.

For example, reporter and analyte nucleic acids may be hybridized to thearray and/or detected simultaneously. Thus, reporter and analyte nucleicacids may be combined prior to hybridization and the array hybridized toboth simultaneously to minimize potential variability in hybridizationconditions. For example, a known amount of labeled reporter and theanalyte nucleic acids can be added to the same hybridization buffer, andthen contacted with one or more arrays simultaneously underhybridization conditions. In another example, a known amount of labeledreporter and analyte nucleic acids are added to the same hybridizationmix, and this buffer aliquoted for the separate hybridization ofdifferent arrays. By storing aliquots of the hybridization mix (e.g.storage at −20° C. or −70° C.), different arrays may be hybridized atdifferent times with approximately the same amounts of the mix.

The term “target” refers to a sample to be analysed. Said target maycomprise the analyte and/or the reporter.

Another feature of reporters added to the analyte in a sample is thatthe concentration and/or amount of the reporter is known.

At the moment of adding the reporter to the sample, the reporter shouldbe structurally as similar as possible to the analyte in the sample.Hence, the reporter can be, e.g. nucleic acids (both DNA and RNA),peptides, polypeptides, proteins, antibodies, carbohydrates, and/orsmall biomolecules. For example, when the analyte is RNA that isconverted to cDNA, then the reporter should preferably be also RNA. Inother words, the structure of the reporter molecule should be as similaras possible to that of the analyte in order to maximally imitate thebinding, e.g. hybridization, of the analyte to, e.g. target nucleicacid. Reporter nucleic acids may be the same length, shorter or longerthan their corresponding internal reference sequences on the array oranalyte nucleic acid in the sample (if present).

However, each reporter nucleic acid should have a least partialcomplementarity to its corresponding internal reference nucleic acid. Inaddition, the reporter nucleic acid should have structural andhybridization characteristics very similar to its corresponding analytenucleic acid, e.g. it should have similar hybridization efficiencies,similar kinetics with complementary probe sequences, similar backgroundhybridization with other sequences, etc. For example, where the analyteset of nucleic acids comprises labeled cDNAs reverse transcribed from acontrol set of a representative pool of synthetic RNAs, the reporternucleic acids will also generally be labeled cDNAs reverse transcribedfrom mRNAs, e.g. synthetic mRNAs.

Each internal reference nucleic acid may be the same length as itscorresponding reporter nucleic acid, longer than its correspondingreporter nucleic acid or shorter than its corresponding reporter nucleicacid. In general, the length of each reporter nucleic acid or set ofreporter nucleic acids in a given sample is at least about 25nucleotides, or at least about 50 nucleotides, or at least about 100nucleotides, where the length could be as a long as 2 kb or longer, butwill generally not exceed about 1 kb and more usually will not exceedabout 800 nucleotides.

The reporter nucleic acid may be synthetic nucleic acids or isolatedfrom a biological source. The reporter nucleic acids may be generatedusing any convenient protocol, including reverse transcription protocols(e.g. using AMV or MoMLV reverse transcriptase), bacteriophage RNApolymerase (T7 RNA polymerase, T3 RNA polymerase, etc.) mediatedtranscription, PCR-, NASBA- or Tyras-protocols, oligonucleotidesynthesis protocols (e.g. nucleotide chemistry), and the like. In anembodiment, the reporter nucleic acid sequences are generated using cDNAfragments doned into appropriate expression vectors using a set of arepresentative number of gene specific primers. These cloned cDNAs arethen used to produce RNA control targets using techniques such as PCRand/or bacteriophage RNA polymerase mediated transcription, NASBA orTyras. Of interest are applications in which the gene specific primersused to generate the reporter are the same as the gene specific primersused to generate the analyte nucleic acids is employed.

After synthesis, each reporter nucleic acid is quantitated usingprocedures such as spectrophotometry, fluorescence measurement, etc.Known quantitative amounts of each reporter nucleic acid are mixed withthe sample for sample preparation or, directly, for use in hybridizationassays, as described herein. In another embodiment, the reporter nucleicacids are mixed together in equal molar amounts, at predeterminedratios, at equal weight amounts, etc, where in many embodiments theywill be mixed together in equal weight amounts, such that the amount ofeach individual reporter nucleic acid in the sample is the same as theanalyte nucleic acid in the sample.

Each reporter molecule should bind to its corresponding internalreference with selectivity and sensitivity. A reporter nucleic acid thatselectively binds with its corresponding internal reference nucleic acidis for example at least 10 times, at least 100 times, or at least 1000times more likely to bind with its designated internal reference nucleicacid than to a non-specific nucleic acid, and preferably any othersequence present on the array. Non-specific nucleic acids include thoseof random sequence, coding sequences found in a particular array otherthan the designated internal reference nucleic acid, and codingsequences of non-internal reference sequences specific to the organismfrom which the internal reference nucleic acids are derived.

Reporter nucleic acids of the invention also display sufficientsensitivity upon binding with their designated internal referencenucleic acids. By “sufficient sensitivity” is meant that binding of thereporter nucleic acid is significantly greater than the binding ofbackground nucleic acids of random sequence, where the strength ofbinding is for example at least 10 times, at least 100 times, or atleast 500 times greater than the recognition of background nucleic acidsof random sequence. In many embodiments, the nucleotide sequences of thesubject reporter nucleic acids are chosen with algorithms, where suchalgorithms are described in detail in PCT publication WO 97/10365 andPCT/US96/14839, the disclosures of which are herein incorporated byreference.

A wide variety of different molecules can be immobilized on thesubstrate of the present arrays. Similarly, the present methods areapplicable to a wide variety of different molecules or receptors thatmay be placed on the substrate of the arrays. The methods and arrays areparticularly exemplified herein in terms of polynucleotides immobilizedon a substrate, but they are equally applicable to other types ofmolecules. For example, one of skilled in the art could easily adapt thepresent methods and arrays to apply to other nucleic acids (both DNA andRNA), peptides, polypeptides, proteins, antibodies, carbohydrates, smallbiomolecules (e.g. drug candidates), or any other types of molecule thatcan be immobilized on a substrate by any method.

The terms “predefined region” or “spot” are used interchangeablythroughout the present invention. The latter terms relate toindividually, spatially addressable positions on an array.

In the present invention, a binding substance is immobilized on thesubstrate at a spatially predefined region, i.e. at a particular spot.The binding substance comprises at least a receptor and a predeterminedamount of an internal reference. The binding substance does not refer toor preclude a linking between the receptor and the internal reference.

For example, the receptor and the internal reference are separatemolecules

In this regard, the term “receptor” refers to any molecule stablyassociated with a substrate which corresponds to a target molecule ofinterest or analyte in a sample, if present. Receptors are not randommolecules, but are predefined.

The term “internal reference” refers to any molecule stably associatedwith a substrate which corresponds to a reporter molecule. The reportermolecule is designed to specifically bind or attach to the internalreference.

The internal references are structurally as similar as possible to thereceptors that are employed in the assays, e.g. both sets of internalreferences and receptors are nucleic acids. In other words, thestructure of the internal reference should be similar to that of thereceptor in order to maximally imitate the binding, e.g. hybridization.

Accordingly, the present invention relates to methods as describedherein, wherein said internal reference comprises nucleic acids,polynucleic acids or (poly)peptides or chemical compounds.

Accordingly, the present invention relates to methods as describedherein, wherein said reporter molecule comprises polynucleic acids or(poly)peptides or chemical compounds.

A critical feature of the arrays of the invention is the predeterminedamounts of the receptors and the internal reference. It will beappreciated by the man skilled in the art that the receptor and theinternal reference may be a hybrid, i.e. the receptor and the internalreference are covalently bound to each other, or the receptor and theinternal reference reside on the same molecule, e.g. a fusion protein.For example, a nucleic acid containing two regions, e.g. a hybrid, ofwhich one region, i.e. internal reference, corresponds to the reporter,while another region, i.e. receptor, corresponds to the analyte. In caseof a hybrid, the amount of the internal reference correlates directly tothe amount of the receptor.

Accordingly, the present invention relates to a method as describedherein, wherein each binding substance immobilized onto said substratecomprises at least 1% to at most 99% of said internal reference.

Accordingly, the present invention relates to a method as describedherein, said each binding substance immobilized onto said substratecomprises the same predetermined amount of said internal reference.

Non-receptor sequences, e.g. control nucleic acids, on the array may nothave a target or corresponding nucleic acid in the analyte or reporterset, e.g. array sequences such as orientation sequences, negative andpositive control sequences, etc. that may be present on an array.

The term “nucleic acid” as used herein means a polymer composed ofnucleotides, e.g. deoxyribonucleotides or ribonucleotides. The terms“ribonucleic acid” and “RNA” as used herein means a polymer composed ofribonucleotides. The terms “deoxyribonucleic acid” and “DNA” as usedherein means a polymer composed of deoxyribonucleotides. The term“oligonucleotide” as used herein denotes single stranded nucleotidemultimers of from about 10 to about 100 nucleotides in length. The term“polynucleotide” as used herein refers to single or double strandedpolymer composed of nucleotide monomers of from about 10 to about 100nucleotides in length, usualy of greater than about 100 nucleotides inlength up to about 1000 nucleotides in length.

The microarrays of the present invention may be of any desired size,from two spots to 10⁶ spots or even more. The upper and lower limits onthe size of the substrate are determined solely by the practicalconsiderations of working with extremely small or large substrates.

For a given substrate size, the upper limit is determined only by theability to create and detect the spots in the microarray. The preferrednumber of spots on a microarray generally depends on the particular useto which the microarray is to be put. For example, sequencing byhybridization will generally require large arrays, while mutationdetection may require only a small array. In general, microarrayscontain from 2 to about 10⁶ spots, or from about 4 to about 10⁵ spots,or from about 8 to about 10⁴ spots, or between about 10 and about 2000spots, or from about 20 to about 200 spots.

Furthermore, not all spots on the microarray need to be unique. Indeed,in many applications, redundancies in the spots are desirable for thepurposes of acting as internal controls.

A variety of techniques have been described for synthesizing and/orimmobilizing arrays of polynucleotides, including in situ synthesis,where the polynucleotides are synthesized directly on the surface of thesubstrate (see, e.g., U.S. Pat. No. 5,744,305 to Fodor, et al.,) andattachment of pre-synthesized polynucleotides to the surface of asubstrate at discrete locations (see, e.g., WO 98/31836). Additionalmethods are described in WO 98/31836 at pages 41-45 and 47-48, amongother places. The present invention is suitable for use with any ofthese currently available, or later developed, techniques.

Immobilization of pre-synthesized polynucleotides at different spatialaddresses yields an array of polynucleotides whose sequences areidentifiable by their spatial addresses.

In embodiments involving in situ synthesis of polynucleotides, thepolynucleotides are synthesized in their usual manner. The syntheticscheme yields an array of polynucleotides whose sequences areidentifiable by their spatial addresses.

While the above method contemplates labeling the last nucleotide of thepolynucleotide, those of skill in the art will appreciate that otherpositions, or additional positions, could be similarly labeled toprovide information about the proportions of truncated polynucleotidessynthesized. In these embodiments, the labels used at the various stepsshould be distinguishable from one another.

Moreover, while the in situ synthesis method is described utilizingphosphoramidite reagents, it will be recognized that other reagentsutilizing other synthesis strategies can also be employed, and incertain circumstances may be preferable, depending on the stability ofthe chosen label to the synthesis conditions. Non-limiting examples ofsuitable chemistries and reagents are described, for example inOligonucleotide Synthesis: A Practical Approach, M. J. Gait, Ed., IRLPress, Oxford, England, 1985.

The composition of the immobilized polynucleotides, e.g. receptors andinternal references, is not critical. The only requirement is that theybe capable of hybridizing to a target nucleic acid of complementarysequence, e.g. reporters and analytes, if any. For example, thepolynucleotides may be composed of all natural or all syntheticnucleotide bases, or a combination of both. Non-limiting examples ofmodified bases suitable for use with the instant invention aredescribed, for example, in Practical Handbook of Biochemistry andMolecular Biology, G. Fasman, Ed., CRC Press, 1989, pp. 385-392. Whilein most instances the polynucleotides will be composed entirely of thenatural bases (A, C, G, T or U), in certain circumstances the use ofsynthetic bases may be preferred.

Moreover, while the backbones of the polynucleotides will typically becomposed entirely of “native” phosphodiester linkages, they may containone or more modified linkages, such as one or more phosphorothioate,phosphoramidite or other modified linkages. As a specific example, oneor more immobilized polynucleotides may be a peptide nucleic acid (PNA),which contains amide interlinkages. Additional examples of modifiedbases and backbones that can be used in conjunction with the invention,as well as methods for their synthesis can be found, for example, inUhlman & Peyman, 1990, Chemical Review 90(4):544-584; Goodchild, 1990,Bioconjugate Chem. 1(3):165-186; Egholm et al., 1992, J. Am. Chem. Soc.114:1895-1897; Gryaznov et al., J. Am. Chem. Soc. 116:3143-3144, as wellas the references cited in all of the above.

As such, the internal reference and receptor nucleic acids may includepolymers of ribonucleotides and deoxyribonucleotides, with theribonucleotide and/or deoxy-ribonucleotides being connected together via5′ to 3′ linkages. Internal reference and receptor nucleic acids of theinvention may be ribonucleic acids, for example sense or antisenseribonucleic acids, full-length or partial fragments of cRNA, full-lengthor partial fragments of mRNA, and/or ribo-oligonucleotides.Alternatively, internal reference and receptor nucleic acids of theinvention may be deoxyribonucleic acids, preferably single-strandedfull-length or fragments of sequences encoding the corresponding mRNAs.The form of the internal reference and receptor nucleic acids should bechosen so that they are complimentary to and form appropriateWatson-Crick hydrogen bonds with reporter and analyte present in asample. For example if analyte sequences in a sample correspond insequence to mRNA, then internal reference and receptor sequences shouldbe complementary, e.g. antisense or complementary RNA (cRNA).

As mentioned above, the internal reference and receptor nucleic acidsmay be polymers of synthetic nucleotide analogs. Such internal referenceand receptor nucleic acids may be utilised in certain embodimentsbecause of their superior stability under assay conditions.Modifications in the native structure, including alterations in thebackbone, sugars or heterocyclic bases, have been shown to increaseintracellular stability and binding affinity. Among useful changes inthe backbone chemistry are phosphorothioates; phosphoro-dithioates,where both of the non-bridging oxygens are substituted with sulfur;phosphoroamidites; alkyl phosphotriesters and boranophosphates. A-chiralphosphate derivatives include 3′-O-5′-S-phosphorothioate,3′-S-5′-O-phosphorothioate, 3′-CH₂-5′-O-phosphonate and3′-NH-5′-O-phosphoroamidate. Peptide nucleic acids replace the entireribose phosphodiester backbone with a peptide linkage. Locked nucleicacids give additional conformational stability of sugar moiety due toadditional bonds between 2′-carboxyl and 5′ carboxyl or 4′-carboxylgroups of deoxyribose. Sugar modifications are also used to enhancestability and affinity. The a-anomer of deoxyribose may be used, wherethe base is inverted with respect to the natural p-anomer. The 2′-OH ofthe ribose sugar may be altered to form 2′-O-methyl or 2′-O-allylsugars, which provides resistance to degradation without comprisingaffinity. Modification of the heterocyclic bases that find use in themethod of the invention are those capable of appropriate base pairing.Some useful substitutions include deoxyuridine for deoxythymidine;5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine fordeoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycitidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

Examples of non-naturally occurring bases that are capable of formingbase-pairing relationships include, but are not limited to, aza- anddeaza-pyrimidine analogues, aza- and deaza-purine analogues, and otherheterocyclic base analogues, wherein one or more of the carbon andnitrogen atoms of the purine and pyrimidine rings have been substitutedby heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and thelike.

The immobilized polynucleotides may be as few as four, or as many ashundreds, or even more, nucleotides in length. Contemplated aspolynucleotides according to the invention are nucleic acids that aretypically referred to in the art as oligonucleotides and also thosereferred to as nucleic acids. Thus, the arrays of the present inventionare useful not only in applications where target nucleic acids arehybridized to immobilized arrays of relatively short (such as, forexample, having a length of approximately 6, 8, 10, 20, 40, 60, 80, or100 nucleotides) probes, but also in applications where relatively shortprobes are hybridized to arrays of immobilized nucleic acids.

The polynucleotides of the array can be of any desired sequence. In apreferred embodiment, they can comprise all possible polynucleotides ofa given length N, which would result in an array of 4^(N) uniqueelements. For all polynucleotides of, for example, 6 bases in length,the sequences would comprise an array with 4096 unique elements.

Alternatively, the polynucleotides can make up the “universal set” forsequencing a nudeic acid, as discussed in WO 98/31836, particularlypages 27-29.

In an alternative embodiment, the set of polynucleotides may correspondto particular mutations that are to be identified in a known sequence.For example, if a particular nucleic acid is known to contain anunidentified mutation at a particular position, then the mutatedposition can be identified with an array of eight polynucleotides, threecorresponding to the three possible substitutions at that position, onecorresponding to the deletion of the base at that position, and fourcorresponding to the insertion of the four possible bases at thatposition. Alternatively, for a known gene that may contain any ofseveral possible identified mutations, the array can comprisepolynucleotides corresponding to the different possible mutations. Thisembodiment is, for instance, useful for genes like oncogenes and tumorsuppressors, which frequently have a variety of known mutations indifferent positions. Using arrays facilitates determining whether or notthese genes contain mutations by allowing simultaneous screening withpolynucleotides corresponding to each of these different positions.

In another alternative embodiment, each spot of the array can comprise amixture of polynucleotides of different sequences. These mixtures maycomprise degenerate polynucleotides of the structure Nx By Nz, wherein Nrepresents any of the four bases and varies for the polynucleotides in agiven mixtures, B represents any of the four bases but is the same foreach of the polynucleotides in a given mixture, and x, y, and z areintegers.

Arrays comprising this type of mixture are useful in, for example,sequencing by hybridization. Alternatively, the spots may comprisemixtures of polynucleotides that correspond to different regions of aknown nucleic acid; these regions may be overlapping, adjacent, ornonadjacent. Arrays comprising these types of mixtures are useful in,for example, identifying specific nucleic acids, including those fromparticular pathogens or other organisms. Both types of mixtures arediscussed in WO 98/31836, particularly at pages 123-128.

The polynucleotides intended for receptors can be isolated frombiological samples, generated by PCR-, NASBA-, Tyras-reactions or othertemplate-specific reactions, or made synthetically. Methods forisolating polynucleotides from biological samples and/or PCR-, Tyras-,NASBA-reactions are well-known in the art, as are methods forsynthesizing and purifying synthetic polynucleotides. Probes isolatedfrom biological samples and/or PCR- Tyras-, NASBA-reactions may,depending on the desired mode of immobilization, require modification atthe 3′- or 5′-terminus, or at one or more bases, as will be discussedmore thoroughly below. Moreover, since the polynucleotide must becapable of hybridizing to a target nucleic acid, if not already singlestranded, it should preferably be rendered single stranded, eitherbefore or after immobilization on the substrate.

The polynucleotides can be immobilized on the substrate using a widevariety of techniques. For example, the polynucleotides can be adsorbedor otherwise non-covalently associated with the substrate (for example,immobilization to nylon or nitrocellulose filters using standardtechniques); they may be covalently attached to the substrate; or theirassociation may be mediated by specific binding pairs, such as biotinand streptavidin.

In order to effect covalent attachment, the substrate must first beactivated, i.e., treated so as to create reactive groups on or withinthe substrate that can react with a reactive group on the polynucleotideto form a covalent linkage. Those of skill in the art will recognizethat the desired reactive group will depend on the chemistry used toattach the polynucleotides to the substrate and the composition of thesubstrate. Typical reactive groups useful for effecting covalentattachment of polynucleotides to substrates include hydroxyl, aldehyde,sulfonyl, amino, epoxy, isothiocyanate and carboxyl groups; however,other reactive groups as will be apparent to those having skill may alsobe used and are also included within the scope of the invention.

For a review of the myriad techniques that can be used to activate thesubstrates with suitable reactive groups, see Wiley Encyclopedia ofPackaging Technology, 2d Ed., Brody & Marsh, Ed., “Surface Treatment,”pp. 867-874. John Wiley & Sons (1997), and the references cited therein(hereinafter “Surface Treatment”). Chemical methods suitable forgenerating amino groups on silicon oxide substrates are described inAtkinson & Smith, “Solid Phase Synthesis of Oligodeoxyribonucleotides bythe Phosphite Triester Method,” In: Oligonucleotide Synthesis: APractical Approach, M J Gait, Ed., 1984, IRL Press, Oxford, particularlyat pp. 45-49 (and the references cited therein); chemical methodssuitable for generating hydroxyl groups on silicon oxide substrates aredescribed in Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026(and the references cited therein); chemical methods for generatingfunctional groups on polymers such as polystyrene, polyamides andgrafted polystyrenes are described in Lloyd-Williams et al., 1997,Chemical Approaches to the Synthesis of Peptides and Proteins, Chapter2, CRC Press, Boca Raton, Fla. (and the references cited therein).

It is contemplated that in general the binding substance is covalentlybound to the substrate. This minimises loss of the binding substancefrom the substrate. Covalent binding of an organic compound to a metaloxide is well known in the art, for example using the method describedby Chu. C. W., et al. (J. Adhesion Sci. Technol., 7, pp. 417-433, 1993)and Fadda, M. B. et al (Biotechnology and Applied Biochemistry, 16, pp.221-227, 1992). Further, after activation of a metal oxide support by asilanating agent and binding of the biomolecules, a number ofamino-groups of said silanating agent can still be present as unloadedamino-groups. This may result in unwanted interactions of saidamino-groups with various substances present in the medium in which theloaded support is used, resulting in high background signals. Theunloaded amino-groups can be removed from the support without affectingthe loaded part of the support by subsequently treating the loadedsupport with an acidic solution. Similarly, an activated and loadedsupport may be treated with a basic or neutral solution, provided thatthe method is not used for derivatization of aluminiumoxidenanoparticles aminated with (3-aminopropyl)-triethoxysilane, wherein thebasic solution further contains a large excess ofN-acetylhomocysteinelactone. In this regard, the European patentapplication PCT/EP00/07736 is exemplary, and is specificallyincorporated in the present invention.

Those of skill in the art will recognize that in embodiments employingcovalent attachment, the covalent bond formed between the polynucleotideand the substrate must be substantially stable to the various conditionsunder which the array will be assayed, to avoid loss of polynucleotideduring the assay. One such stable bond is the phosphodiester bond, whichconnects the various nucleotides in a polynucleotide, and which can beconveniently formed using well-known chemistries (see, e.g.,Oligonucleotide Synthesis: A Practical Approach, 1984, supra). Otherstable bonds suitable for use with hydroxyl-activated substrates includephosphorothioate, phosphoramidite, or other modified nucleic acidinterlinkages. For substrates modified with amino groups, the bond couldbe a phosphoramidate, amide or peptide bond. When substrates areactivated with epoxy functional groups, a stable C—N bond could beformed. Suitable reagents and conditions for forming such stable bondsare well known in the art. Other stable bonds suitable for use with thearrays of the invention will be apparent to those of skill in the art.

In embodiments in which pre-synthesized polynucleotides are covalentlyattached to the substrate, the polynucleotides may be attached via their3′-terminus, 5′-terminus or by way of a reactive group at one of thebases. Synthesis supports and synthesis reagents useful for modifyingthe 3′- and/or 5′-terminus of synthetic polynucleotides, or forincorporating a base modified with a reactive group into a syntheticpolynucleotide, are well-known in the art and are also commerciallyavailable.

For example, methods for synthesizing 5′-modified polynucleotides aredescribed in Agarwal et al., 1986, Nucl. Acids Res. 14:6227-6245 andConnelly, 1987, Nucl. Acids Res. 15:3131-3139. Commercially availableproducts for synthesizing 5′-amino modified polynucleotides include theN-TFA-C6-AminoModifier, N-MMT-C6-AminoModifier andN-MMT-C12-AminoModifier reagents available from Clontech Laboratories,Inc., Palo Alto, Calif.

Methods for synthesizing 3′-modified polynucleotides are described inNelson et al., 1989, Nucl. Acids Res. 17:7179-7186 and Nelson et al.,1989, Nucl. Acids Res. 17:7187-7194. Commercial products forsynthesizing 3′-modified polynucleotides include the 3′-Amino-ON™.controlled pore glass and Amino Modifier II™ reagents available fromClontech Laboratories, Inc., Palo Alto, Calif.

Other methods for modifying the 3′ and/or 5′ termini of polynucleotides,as well as for synthesizing polynucleotides containing appropriatelymodified bases are provided in Goodchild, 1990, Bioconjugate Chem.1:165-186, and the references cited therein. Chemistries for attachingsuch modified polynucleotides to substrates activated with appropriatereactive groups are well-known in the art (see, e.g., Ghosh & Musso,1987, Nucl. Acids Res. 15:5353-5372; Lund et al., 1988, Nucl. Acids Res.16:10861-10880; Rasmussen et al., 1991, Anal. Chem. 198:138-142; Kato &Ikada, 1996, Biotechnology and Bioengineering 51:581-590; Timofeev etat., 1996, Nucl. Acids Res. 24:3142-3148; O'Donnell et al., 1997, Anal.Chem. 69:2438-2443).

Methods and reagents for modifying the ends of polynucleotides isolatedfrom biological samples and/or for incorporating bases modified withreactive groups into nascent polynucleotides are also well-known andcommercially available. For example, an isolated polynucleotide can bephosphorylated at the 5′-terminus with phosphorokinase and thisphosphorylated polynucleotide covalently attached to an amino-activatedsubstrate through a phosphoramidate or phosphodiester linkage. Othermethods will be apparent to those of skill in the art.

In one convenient embodiment, pre-synthesized polynucleotides, modifiedat their 3′- or 5′-termini with a primary amino group, are conjugated toa carboxy-activated substrate. Chemistries suitable for formingcarboxamide linkages between carboxyl and amino functional groups arewell-known in the art of peptide chemistry (see, e.g., Atherton &Sheppard, Knorr et al., 1989, Tet. Left. 30(15):1927-1930; Bannworth &Knorr, 1991, Tet. Lett. 32(9):1157-1160; and Wilchek et al., 1994,Bioconjugate Chem. 5(5):491-492; Solid Phase Peptide Synthesis, 1989,IRL Press, Oxford, England and Lloyd-Williams et al., ChemicalApproaches to the Synthesis of Peptides and Proteins, 1997, CRC Press,Boca Raton, Fla. and the references cited therein). Any of these methodscan be used to conjugate amino-modified polynucleotides to acarboxy-activated substrate.

Whether synthesized directly on the activated substrate or immobilizedon the activated substrate after synthesis or isolation, thepolynucleotides can optionally be spaced away from the substrate by wayof one or more linkers. As will be appreciated by those having skill inthe art, such linkers will be at least bifunctional, i.e., they willhave one functional group or moiety capable of forming a linkage withthe activated substrate and another functional group or moiety capableof forming a linkage with another linker molecule or thepolynucleotides.

Stretches of nucleotides can be interrupted by one or more linkermolecules that do not participate in sequence-specific base pairinginteractions with a target nucleic acid. The linker molecules may beflexible, semi-rigid or rigid, long or short, charged or uncharged,hydrophobic or hydrophilic, depending on the desired application. Avariety of linker molecules useful for spacing one molecule from anotheror from a solid surface have been described in the art; all of theselinker molecules can be used to space regions of immobilizedpolynucleotides from one another. In an embodiment of this aspect of theinvention, the linker moiety is from one to ten, from one to six,alkylene glycol moieties, e.g. ethylene glycol moieties.

In certain circumstances, such linkers can be used to “convert” onefunctional group into another. For example, an amino-activated substratecan be converted into a hydroxyl-activated substrate by reaction with,for example, 3-hydroxy-propionic acid. In this way, substrate materialswhich cannot be readily activated with a specified reactive functionalgroup can be conveniently converted into an appropriately activatedsubstrate. Chemistries and reagents suitable for “converting” suchreactive groups are well-known, and will be apparent to those havingskill in the art.

Linkers can also be used, where necessary, to increase or “amplify” thenumber of reactive groups on the activated substrate. For thisembodiment, the linker will have three or more functional groups.Following attachment to the activated substrate by way of one of thefunctional groups, the remaining two or more groups are available forattachment of polynucleotides. Amplifying the number of functionalgroups on the activated substrate in this manner is particularlyconvenient when the substrate cannot be readily activated with asufficient number of reactive groups.

Reagents for amplifying the number of reactive groups are well-known andwill be apparent to those of skill in the art. A particularly convenientclass of amplifying reagents are the multifunctional epoxides sold underthe trade name DENACOL™. (Nagassi Kasei Kogyo K. K.). These epoxidescontain as many as four, five, or even more epoxy groups, and can beused to amplify substrates activated with reactive groups that reactwith epoxides, including, for example, hydroxyl, amino and sulfonylactivated substrates. The resulting epoxy-activated substrate can beconveniently converted to a hydroxyl-activated substrate, acarboxy-activated substrate, or other activated substrate by well-knownmethods.

Linkers suitable for spacing biological molecules such aspolynucleotides from solid surfaces are well-known in the art, andinclude, by way of example and not limitation, polypeptides such aspolyproline or polyalanine, saturated or unsaturated bifunctionalhydrocarbons such as 1-amino-hexanoic acid, polymers such aspolyethylene glycol, etc. 1,4-Dimethoxytrityl-polyethylene glycolphosphoramidites useful for forming phosphodiester linkages withhydroxyl groups, as well as methods for their use in nucleic acidsynthesis on solid substrates, are described, for example in Zhang etal., 1991, Nucl. 20 Acids Res. 19:3929-3933 and Durand et al., 1990,Nucl. Acids Res. 18:6353-6359. Other useful linkers are commerciallyavailable.

The nature and geometry of the solid substrate will depend upon avariety of factors, including, among others, the type of array (e.g.,one-dimensional, two-dimensional or three-dimensional) and the mode ofattachment (e.g., covalent or non-covalent). Generally, the substratecan be composed of any material which will permit immobilization of thereceptor, e.g. polynucleotide, and which will not melt or otherwisesubstantially degrade under the conditions used to bind the receptor,e.g. hybridize and/or denature nucleic acids. In addition, wherecovalent immobilization is contemplated, the substrate should beactivatable with reactive groups capable of forming a covalent bond withthe receptor to be immobilized.

A number of materials suitable for use as substrates in the instantinvention have been described in the art. Exemplary suitable materialsinclude, for example, acrylic, styrene-methyl methacrylate copolymers,ethylene/acrylic acid, acrylonitrile-butadienestyrene (ABS),ABS/polycarbonate, ABS/polysulfone, ABS/polyvinyl chloride, ethylenepropylene, ethylene vinyl acetate (EVA), nitrocellulose, nylons(including nylon 6, nylon 6/6, nylon 6/6-6, nylon 6/9, nylon 6/10, nylon6/12, nylon 11 and nylon 12), polycarylonitrile (PAN), polyacrylate,polycarbonate, polybutylene terephthalate (PBT), polyethyleneterephthalate (PET), polyethylene (induding low density, linear lowdensity, high density, cross-linked and ultra-high molecular weightgrades), polypropylene homopolymer, polypropylene copolymers,polystyrene (including general purpose and high impact grades),polytetrafluoroethylene (PTFE), fluorinated ethylene-propylene (FEP),ethylenetetrafluoroethylene (ETFE), perfluoroalkoxyethylene (PFA),polyvinyl fluoride (PVF), polyvinylidene fluoride (PVDF),polychlorotrifluoroethylene (PCTFE), polyethylenechlorotrifluoroethylene(ECTFE), polyvinyl alcohol (PVA), silicon styreneacrylonitrile (SAN),styrene maleic anhydride (SMA), and glass.

Other exemplary suitable materials for use as substrates in the presentinvention include metal oxides. Metal oxides provide a substrate havingboth a high channel density and a high porosity, allowing high densityarrays comprising different first binding substances per unit of thesurface for sample application. In addition, metal oxides are highlytransparent for visible light. Metal oxides are relatively cheapsubstrates that do not require the use of any typical microfabricationtechnology and, that offers an improved control over the liquiddistribution over the surface of the substrate, such aselectrochemically manufactured metal oxide membrane. Metal oxidemembranes having through-going, oriented channels can be manufacturedthrough electrochemical etching of a metal sheet. Metal oxidesconsidered are, among others, oxides of tantalum, titanium, andaluminum, as well as alloys of two or more metal oxides and doped metaloxides and alloys containing metal oxides. The metal oxide membranes aretransparent, especially if wet, which allows for assays using variousoptical techniques. Such membranes have oriented through-going channelswith well controlled diameter and useful chemical surface properties.Patent application EP-A-0 975 427 is exemplary in this respect, and isspecifically incorporated in the present invention.

Accordingly, the present invention relates to a method as describedherein, wherein said microarray is a flow-through microarray.

Accordingly, the present invention relates to a method as describedherein, wherein said substrate is a porous substrate.

Accordingly, the present invention relates to a method as describedherein, wherein said substrate is an electrochemically manufacturedmetal oxide membrane.

Accordingly, the present invention relates to a method as describedherein, wherein said substrate comprises aluminum oxide.

The substrate may be in the form of beads, particles, sheets, ormembranes and may be permeable or impermeable, depending on the type ofarray. For example, for linear or three-dimensional arrays the substratemay consist of bead or particles (such as conventional solid phasesynthesis supports), fibers (such as glass wool or other glass orplastic fibers), glass or plastic capillary tubes, or metal oxidemembranes. For two-dimensional arrays, the substrate may be in the formof plastic or glass sheets in which at least one surface issubstantially flat.

The detection of the reporter is indicative for the presence, amountand/or integrity of the analyte. Thus, it is important that theefficiencies of the binding between analyte and receptor, as well asreporter and internal reference are substantially similar. Similarly, itis important that the detection of complexed analyte and receptor, aswell as complexed reporter and internal reference are substantiallysimilar.

Use of the arrays of the present invention contemplates the use ofreporter polynucleotides and/or analyte nucleic acids that are capableof generating a signal when appropriately bound, e.g. hybridized, to thearray.

The signal generated by the internal reference is measured or determinedby means of a binding reaction, for example, a hybridisation reaction,with a labeled reporter. The signal generated by the binding of thereporter to the internal reference is preferably distinguishable fromthe signal generated by the binding of the analyte to the receptor.

Depending on the particular assay protocol with which the subjectanalyte and reporter nucleic acids are employed, the analyte andreporter nucleic acids may be labeled with the same label, such that theanalyte and reporter cannot be distinguished from one another, or theanalyte and reporter nucleic acids may be differentially labeled, suchthat the two sets are readily and/or simultaneously distinguishable fromeach other.

As such, in certain embodiments, the analyte and reporter nucleic acidsare differentially labeled. By “differentially labeled” is meant thatthe reporter and analyte nucleic acids are labeled differently from eachother such that they can be simultaneously distinguished from eachother. For example, where one has reporter nucleic acids and analytenucleic acids, each reporter nucleic acid in the sample will be labeledwith the same first label and each analyte nucleic acid in the samplewill be labeled with the same second label that is different anddistinguishable from the first label. Likewise, where two different setsof reporter nucleic acids are employed in the method, each reporternucleic acid in the second set will be labeled with a third labeldifferent and distinguishable from both the first and second label.

Virtually any label that produces a detectable, quantifiable signal andthat is capable of being attached to an analyte and/or reporter, e.g.polynucleotides, can be used in conjunction with the arrays of theinvention. Suitable labels include, by way of example and notlimitation, radioisotopes, fluorophores, chromophores, chemiluminescentmoieties, etc. In embodiments where the label is attached to apolynucleotide, the label can be attached to any part of thepolynucleotide, including the free terminus or one or more of the bases.Preferably, the position of the label will not interfere withhybridization, detection or other post-hybridization modifications ofthe labeled polynucleotide. A variety of different protocols may be usedto generate the labeled nucleic acids, as is known in the art, wheresuch methods typically rely on the enzymatic generation of labelednucleic acid using an initial primer and template nucleic acid. Labeledprimers can be employed to generate the labeled target. Alternatively,label can be incorporated into the nucleic acid during first strandsynthesis or subsequent synthesis, labeling or amplification steps inorder to produce labeled target. Label can also be incorporated directlyto mRNA using chemical modification of RNA with reactive labelderivatives or enzymatic modification using labeled substrates.Representative methods of producing labeled target are disclosed in U.S.application Ser. Nos.: 08/859,998; 08/974,298; 09/225,998; thedisclosures of which are incorporated herein by reference.

The reporter polynucleotides or analyte nucleic acids may be labeled,for example, by the labels and techniques described supra.Alternatively, they may be labeled by any other technique known in theart. Preferred techniques include direct chemical labeling methods andenzymatic labeling methods, such as kinasing and nick-translation.

A variety of different labels may be employed, where such labels includefluorescent labels, isotopic labels, enzymatic labels, particulatelabels, etc. For example, suitable labels include fluorochromes, e.g.fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin, 6-carboxyfluorescein (6-FAM), 2′, 7′-dimethoxy4′,5′-dichloro-6-carboxy-fluorescein (JOE), 6-carboxy-X-rhodamine (ROX),6-carboxy-2′, 4′, 7′, 4,7-hexachloro-fluorescein (HEX),5-carboxyfluorescein (5-FAM) or N, N, N′,N′-tetramethyl-6-carboxy-rhodamine (TAMRA), cyanine dyes, e.g. Cy5, Cy3,BODIPY dyes, e.g. BODIPY 630/650, Alexa542, etc. Suitable isotopiclabels include radioactive labels, e.g. ³²P, ³³P, ³⁵S, ³H. othersuitable labels include size particles that possess light scattering,fluorescent properties or contain entrapped multiple fluorophores. Thelabel may be a two stage system, where the target DNA is conjugated tobiotin, haptens, etc. having a high affinity binding partner, e.g.avidin, specific antibodies, etc. The binding partner is conjugated to adetectable label, e.g. an enzymatic label capable of converting asubstrate to a chromogenic product, a fluorescent label, an isotopiclabel, etc. Similarly, the detection of the binding between analyte andreceptor, as well as the reporter and the internal reference can beindirect. In the present invention, indirect detection relates to thedetection of a possible interaction between analyte and receptor or thereporter and the internal reference, in which either the analyte andreceptor, and/or the reporter and the internal reference are notlabeled. For example, the present invention relates to a sandwich assay,in which analyte and the reporter are antibodies, and wherein theanalyte and the reporter are from different species.

It is contemplated that the man skilled within the art will be able toadapt the array format of the present invention to his specific needs.For example, the skilled man may adapt the array format such that thebinding of the analyte to the receptor can be detected directly, whilethe binding of the reporter to the internal reference is detectedindirectly. Any combination of labels, e.g. first and second labels,first, second and third labels, etc., may be employed for the reportersets and analyte in a sample, provided the labels are distinguishablefrom one another. Examples of distinguishable labels are well known inthe art and include: two or more different emission wavelengthfluorescent dyes, like Cy3 and Cy5, or Alexa 542 and Bodipy 630/650; twoor more isotopes with different energy of emission, like ³²P and ³³P;labels which generate signals under different treatment conditions, liketemperature, pH, treatment by additional chemical agents, etc., andlabels which generate signals at different time points after treatment.

Using one or more enzymes for signal generation allows for the use of aneven greater variety of distinguishable labels based on differentsubstrate specificity of enzymes, e.g. alkaline phosphatase/peroxidase.

Accordingly, the present invention relates to a method as describedherein, wherein the reporter comprises a label.

Accordingly, the present invention relates to a method as describedherein, wherein the analyte is labeled.

Accordingly, the present invention relates to a method as describedherein, wherein the label of the analyte and/or reporter is of theenzymatic, fluorescent, phosphorescent or radioactive type.

Accordingly, the present invention relates to a method as describedherein, wherein the label of the analyte differs from the label of theinternal reference.

Accordingly, the present invention relates to a method as describedherein, wherein the label of the analyte is Texas red, and the label ofthe internal reference is fluorescein.

Accordingly, the present invention relates to a method as describedherein, wherein the label of the internal reference is Texas red, andthe label of the analyte is fluorescein.

In embodiments employing in situ synthesis, a preferred label is afluorescently labeled nucleic acid synthesis reagent, such as a labelednucleoside phosphoramidite. The position at which the fluorophore isattached to the nucleoside phosphoramidite will depend on whether thelabel will be added at the terminal or internal nucleotides of thenascent polynucleotides. When a terminal label is desired, thefluorophore can be conveniently attached to the 5′-hydroxyl. Wheninternal labels are desired, the flurophore is preferably attached tothe base, optionally by way of a linker. Methods suitable for makingfluorescently-labeled phosphoramidite synthesis reagents are well-knownin the art, and are described, for example, in Goodchild, 1990, supra.

The present invention contemplates that molecules used herein, can bemolecular beacons. For example, the receptor and/or the internalreference can be molecular beacons, in which case the analyte and/orreporter are target nucleic acids, respectively. Alternatively, thereporter and/or the analyte can be molecular beacons, in which case theinternal reference and/or receptor are target nucleic acids,respectively. Another possibility is that the reporter and the receptorare molecular beacons, in which case the internal reference and/oranalyte are target nucleic acids, respectively. Molecular beacons arehairpin-shaped molecules with an internally quenched fluorophore whosefluorescence is restored upon binding to a target nucleic acid. The loopportion of the molecular beacon is complementary to a target, whereasthe stem is formed by the annealing of complementary arm sequences. Afluorescent label and a quenching group are attached at the respectiveends of the molecular beacon. The stem holds these two groups in closeproximity to each other, causing the fluorescence of the fluorophore tobe quenched by energy transfer. The quenching group is a non-fluorescentchromophore and emits the energy that it receives from the fluorophoreas heat. When the molecular beacon encounters a target molecule, themolecular beacon forms a hybrid that is more stable than the stem. Thus,the molecular beacon undergoes a spontaneous conformationalreorganization that forces the stem apart, and causes the fluorophoreand the quencher to move away from each other, leading to therestoration of fluorescence which can be detected. Disclosures by Tyagiand Kramer (1996; Nature Biotechnology 14:303-308) and van Beuningen etal. (Proceedings of SPIE vol. 4264 (2001) 66-71) are exemplary in thisrespect, and specifically incorporated in the present invention. Thequenching moiety of the molecular beacon can be combined with a numberof different fluorophores. For example, if two fluorophores areemployed, these fluorophores may be different, e.g. the fluorophore ofthe receptor may differ from the fluorophore of the internal reference.

The present invention contemplates the use of nucleic acid aptamers fordetection. An aptamer is an oligonucleotide with a unique sequence thatfolds into a unique secondary and tertiary structure that, inconsequence, present a unique binding surface to its ligands. In thisregard, the present invention relates also to aptamer beacons.

Molecular and aptamer beacons may be employed in indirect detection,i.e. detection with a molecular or aptamer beacon of an analyte bound toa receptor and/or of a reporter bound to its internal reference.

For embodiments employing immobilization of pre-synthesizedpolynucleotides, a preferred label is a labeled polynucleotide. Theprimary sequences of the labeled and unlabeled polynucleotides at aparticular spot may be the same or different. In fact, the same labeledpolynucleotide may be used at each spot in the array. The onlyrequirement is that the polynucleotide reagents deposited at each spotin the array be “spiked” with substantially the same proportion oflabeled polynucleotide.

In an embodiment, the same mixture of labeled polynucleotides is used tospike the polynucleotide reagent deposited at each spot. Using the samemixture of labeled polynucleotides at each spot ensures that the labelsat different spots do not induce sequence-specific anomalies inhybridization assays, i.e., it ensures that the labels at each arrayspot interact similarly with a target nucleic acid in hybridizationassays. Moreover, use of the same label at each spot reduces the numberof labeled polynucleotides that need to be prepared.

The amount of label used to “spike” the polynucleotide reagent to bedeposited at a particular spot is not critical for success. However, theamount used should be sufficient to produce a detectable signal whichdoes not result in a loss of dynamic range when the array is used in anassay.

For use in a hybridization array, the background signals from apolynucleotide array according to the invention are quantified andrecorded. The mode of detection will depend on the nature of the label.For fluorescent labels, the background signals can be convenientlyquantified by scanning the array with a confocal camera or with a CCDcamera, as is well-known in the art.

The array is contacted with a reporter and analyte nucleic acid, whichmay be labeled or unlabeled, depending on the particular array format,under conditions which discriminate between perfectly complimentaryhybrids and hybrids containing one or more mismatches. The actualhybridization conditions used will depend upon, among other factors, theG+C content of the sequence of interest and the lengths of theimmobilized polynucleotides comprising the array. Hybridizationconditions useful for discriminating between perfect compliments andmismatches for a variety of hybridization arrays have been described inthe art. For example, hybridization conditions useful for discriminatingcomplimentary and mismatched hybrids in a variety of applications aredescribed in U.S. Pat. No. 5,525,464 to Drmanac et al., WO 95/09248 andWO 98/31836. A detailed discussion of the theoretical and practicalconsiderations involved in determining hybridization conditions, andincluding a discussion of the advantages of low-temperature washingsteps, may be found in WO 98/31836, particularly pages 50-62. Additionalguidance may be found in Harmes and Higgins, Nucleic Acid Hybridization:A Practical Approach, 1985, IRL Press, Oxford, England.

As mentioned above, in practicing the subject methods the analyte andreporter nucleic acids are hybridized to an array, where the targetcomprising analyte and reporter nucleic acids may be hybridized to thesame array or different arrays, where when the analyte and reporternucleic adds are hybridized to different arrays, all of the differentarrays may at least share common arrays, spots or binding substances ofreceptor and/or internal reference nucleic acids, e.g. they will beidentical with respect to their receptor and/or internal referencenucleic acids.

In the above embodiments where the analyte and reporter nucleic acidsare hybridized simultaneously to a given array, labeled analyte andreporter nucleic acids are premixed or pooled prior to contact with thearray. In an embodiment, mixtures of analyte and reporter nucleic acidshave amounts of the analyte and reporter nucleic acids which aresufficient to generate signals that are at least 1.5 fold, usually atleast 3 fold and more usually at least 5 fold higher than backgroundsignals observed with the array. The relative amounts of the analyte andreporter nucleic acids in the mixture are selected to be sufficient toallow reliable detection of the test sequences complimentary to therespective receptor and internal reference nucleic acid while at thesame time allowing complete binding of the reporter nucleic acids with anofold excess of unbound reporter nucleic acid on the array. The amountof reporter nucleic acid present in the mixture is usually determined byavailable amount of sample and sensitivity of technology employed in aparticular protocol. For example, the amount of reporter nucleic acidpresent in the mixture ranges from about 0.01-100 μg of nucleic acid,e.g. cDNA, and more usually from about 0.1-10 μg of nucleic acid, e.g.cDNA. In many embodiments, the amount of reporter nucleic acid employedin the hybridization protocol is about the same or less than the amountof analyte nucleic acid that is employed, where less than typicallymeans 10 fold less, usually 100 fold less and more usually 1000 foldless. Of interest are mixtures of labeled nudeic acids that provide foran intensity of signal from each probe nucleic acid in the controldetection channel that ranges from about 0.001 to 0.1%, usually fromabout 0.001 to 0.01% abundance level.

The reporter and analyte nucleic acids are hybridized to the array(s) bycontacting the analyte and reporter nucleic acids with the array(s)under hybridization conditions. By “hybridization conditions” is meantconditions sufficient to promote Watson-Crick hydrogen bonding to occurbetween the target and probe nucleic acids. The hybridizationconditions, such as hybridization time, temperature, wash buffers used,etc. can be altered to optimize the efficient and specific binding ofthe target sequences. Test target nucleic acids having sequencesimilarity to the probes may be detected by hybridization under lowstringency conditions, for example, at 50° C. and 6×SSC (0.9 M sodiumchloride/0.09 M sodium citrate, 1% SDS) and remain bound when subjectedto washing at 55° C. in 1×SSC (0.15 M sodium chloride/0.015 M sodiumcitrate, 1% SDS). Test target sequences with sequence identity may bedetermined by hybridization under stringent conditions, for example, at60° C. or higher and 6×SSC (15 mM sodium chloride/01.5 mM sodiumcitrate, 1% SDS). For example, the analyte and reporter nucleis acidshave a region of substantial identity to the provided receptor andinternal reference sequences on the array, respectively, and bindselectively to their respective receptor and internal referencesequences under stringent hybridization conditions. Other suitablehybridization conditions for various nucleic acid pairs are well knownto those skilled in the art and reviewed in Sambrook et al., 1989 (seeinfra), and in PCT WO 95/21944, the disclosure of which is hereinincorporated by reference.

Analysis of the differences in signal generated by two or more sourcesmay be carried out by using multiple arrays with the same or similarreceptor and internal reference compositions, each array for each set ofanalyte and reporter nucleic acids. Each array is then hybridized withlabeled reporter target nucleic acids and labeled analyte nucleic acids.For instance, the labeling efficiency and amount of analyte sequencesand reporter sequences is approximately equivalent between arrays, e.g.an equal amount of labeled analyte nucleic acids is used to hybridize toeach array. This is not essential, however, since hybridization of theset of labeled reporter nucleic acids functions as an independentinternal control for each probed array.

Levels of hybridization of reporter RNA to the binding substances can bestandardized by comparing the hybridization signal of the reporter withinternal reference sequences on each array.

Differences in hybridization of the predefined reporter sequences to thepredefined internal references allows a comparison of relativehybridization levels between arrays

Following hybridization, non-hybridized labeled nudeic acid is removedfrom the substrate, conveniently by washing, generating a pattern ofhybridized nucleic acid on the substrate surface. A variety of washsolutions and protocols are known to those of skill in the art and maybe used. See Sambrook, Fritsch & Maniatis, Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Press) (1989).

If the analyte and/or reporter is labeled, the array can be scanned orotherwise analyzed for detectable assay signal, and the signal from eachlabeled spot, or alternatively from all spots, quantified. Only thosespots where binding, e.g. hybridization, occurred will produce adetectable assay signal. The resultant hybridization patterns of labelednucleic acids may be visualized or detected in a variety of ways, withthe particular manner of detection being chosen based on the particularlabel of the target nucleic acid, where representative detection meansinclude scintillation counting, autoradiography, fluorescencemeasurement, colorimetric measurement, light emission measurement, lightscattering and the like (see above).

Following detection, determination or visualization, the binding, e.g.hybridization, patterns generated by analyte and reporter, for exampleanalyte and reporter nucleic acids, may be compared to identifydifferences between the signals. Where arrays in which each of thedifferent receptor corresponds to a known gene are employed, differencesin signal intensity can be related to a different analyte concentrationof a particular gene.

The comparison of the intensity of the signal resulting from the bindingof an analyte nucleic acid to a receptor can be compared to theintensity of the signal resulting from the binding of the correspondingreporter sequence to the internal reference sequence, and themeasurement converted to a relative quantitative nucleic acidconcentration for that analyte sample. The relative quantitative nucleicadd levels of the analyte can be compared within and between arrays toidentify, determine or confirm differential expression of genes inparticular samples.

If each spot in the array contains the same quantity of immobilizedpolynucleotide, in theory, the intensity of the assay signal at eachspot will be proportional to the extent of hybridization at that spot.For example, spots containing perfectly complementary hybrids areexpected to produce more intense assay signals than spots containingmismatched hybrids. In practice, however, differences in signalintensities between different spots may instead be due to differences inthe amounts of polynucleotide immobilized at the respective spots oramounts of analytes due to sample preparation.

Because each spot in the arrays of the invention contains an amount ofan internal reference proportional to the amount of receptor immobilizedat the particular spot, the assay signals obtained from the arrays ofthe invention can be normalized. As a consequence, signal intensitiesfrom spots within a single array, within spots, or across multiplearrays, can be directly compared, without regard to the fidelity of theparticular array synthesis or the sample preparation.

The method by which the signals are normalized will depend upon whetherthe reporter or background signals are the same as the assay signals,such as where the reporter and analyte are labeled with the samefluorophore. In this embodiment, a normalized signal of a particularspot is defined by (Ia-Ib)/Ib, where Ia is the intensity of the assaysignal of the spot (e.g. intensity of the spot after hybridization) andIb is the intensity of the background signal of the spot (e.g. theintensity of the spot before hybridization).

In embodiments where the reporter and assay signals are different, e.g.where the reporter and the analyte are differently labeled, e.g. withdifferent fluorophores, the normalized signal for a spot is described byIa/Ib, where Ia is the intensity of the assay signal of the analyte andlb is the intensity of the reporter signal of the same spot.

Accordingly, the present invention relates to a method for thenormalization of an array comprising the steps of:

-   -   (i) immobilizing onto said array a binding substance comprising        a receptor and a predetermined amount of an internal reference,        and,    -   (ii) determining the signal generated by said internal reference        by means of a reporter molecule which selectively binds to said        internal reference.

While the array is illustrated utilizing labeled analyte and reporternucleic acids, those of skill in the art will recognize that the arraysof the invention are also useful in assays employing unlabeled targetnucleic acids. The only requirement is that some component of theparticular assay generate a detectable signal at spots where binding,e.g. hybridisation, occurs.

The subject methods find use in, among other applications,standardization of differential gene expression assays. Thus, one mayuse the subject methods in the differential expression analysis of: (a)diseased and normal tissue, e.g. neoplastic and normal tissue, (b)different tissue or tissue types; (c) developmental stage; (d) responseto external or internal stimulus; (e) response to treatment; and thelike. The methods of the subject invention therefore find use in broadscale expression screening for drug discovery, diagnostic and research,such as the effect of a particular active agent on the expressionpattern of genes in a particular cell, where such information can beused to reveal drug toxicity, carcinogenicity, etc., environmentalmonitoring, disease research and the like. A number of different taskscan be accomplished with the subject invention, which tasks include, butare not limited to: detecting relative hybridization of targetsequences, calibrating a hybridization assay, harmonizing data betweenhybridization assays, and testing reagents used in a hybridizationassay. The subject methods in which control and test sets of targetnucleic acids are employed can also be used in the generation of geneexpression databases, as the data generated from the subject methods arerelative quantitative, reflect relative RNA concentration rather thanintensity of signal, and are independent of the type of array. Each ofthese different aspects of the invention is discussed separately below.

The methods of the present invention are useful in detecting relativelevels of hybridization of different genes in a sample by providing aset of internal hybridization controls, i.e. the reporter. Since thereporter nucleic acids are of a known sequence, in a known quantity, andof a known specific activity (where in an exemplary embodiment thereporter and analyte are labeled with the same specific activity), thelevel of hybridization of the reporter nucleic acids can be used todetermine the level of expression of each gene in a test sample based onits level of binding to a receptor sequence. The provision that eachsample has its own internal control (reporter) also allows for thedetection of potential expression differences between samples anddifferences in binding affinities between receptor sequences, both on asingle array and between arrays. Thus, the intensity level ofhybridization of a reporter sequence can be used to calculate theexpression level of a gene in a sample based upon the intensity of theanalyte hybridization to the corresponding receptor sequence.

The methods of the subject invention also find use in the calibration ofhybridization assays. Using known concentrations of receptor nucleicacid, analyte nucleic acids, internal reference nucleic acids andreporter nucleic acids allows one to optimize the hybridizationconditions for a particular use, such as increasing stringency to allowbetter detection of nucleic acids with some level of sequence homology(e.g. differential expression between genes from a single family oralternative splice forms for the same gene). The use of the internalstandards of the method of the subject invention allows hybridization,labeling procedures, and the like to be optimized for a particular use,which is especially valuable for standardization of large scale ofhybridization assays, such as high throughput screening of biologicalsamples. Optimization thus means that one can change hybridizationconditions in order to achieve maximal intensity of specifichybridization signals with complimentary probe sequences and minimallevel of non-specific hybridization with non-complementary probesequences.

The methods of the subject invention also find use in the harmonizationof data between hybridization assays, thus allowing for a directcomparison of expression levels despite potential differences due tovariables such as differences in hybridization conditions, differencesin sample preparation and even between different types of arrays,differences in quality and performance within and between differentarrays, differences in specific activity of the labeled targetsequences, and the like. Because each hybridization assay has itsinternal control for at least a subset of the probe sequences on thearray, the data can be compared using ratios of the intensity of thereporter nucleic acids and the intensity of the analyte nucleic acids.Thus, the use of simple mathematical formulations to correct fordifferences between assays allows the levels of gene expression in thesedifferent assays to be adjusted to the same level and then compared in abiologically relevant fashion.

The methods of the present invention are also useful in determining theefficacy of hybridization reagents. Such reagents may be, for example,new reagents, e.g. different buffer solutions for prehybridization andhybridization, or established reagents, e.g. a new batch of a known,commercially available reagent. The internal control of the methods ofthe subject invention provide for two levels of quality assurance upontesting the reagents, basically providing an extra control fordetermining the efficacy of a reagent in a single hybridization.Efficiency means maximum specific signal with minimal level ofnon-specific signal and background binding to solid surface. Otherparameters such as temperature, buffer composition, length ofhybridization and/washing times, etc., may be optimized usingcalibration controls. Also, the same calibration reporter nucleic acidscan be used routinely to test and calibrate detection equipment toexpected level intensity of signals, thus limiting variability due tofunctionality of the equipment; variation due to data generated indifferent labs, or at different times, or even using different types ofarrays.

Accordingly, the present invention relates to a method as describedherein for use in expression profiling assay, genotyping, sequencedetermination by hybridization, gene quantitation, gene abnormalityanalysis (Multiplex Amplifiable Probe Hybridisation, MAPH), PCR, NASBA,or TYRAS.

Accordingly, the present invention relates to the use of an array fornormalisation of analyte variation, wherein said array comprises asubstrate with predefined regions, wherein each binding substanceimmobilized at a predefined region of said substrate comprises areceptor and a predetermined amount of an internal reference, whereinthe signal generated by said internal reference is determined by meansof a reporter molecule and wherein said reporter molecule selectivelybinds to said internal reference.

Accordingly, the present invention relates to the use of an array in amethod as described herein.

Accordingly, the present invention relates to an array for use in amethod as described herein, wherein said array comprising a substratewith predefined regions, wherein each binding substance immobilized at apredefined region of said substrate comprises a receptor and apredetermined amount of an internal reference, wherein the signalgenerated by said internal reference is determined by means of areporter molecule and wherein said reporter molecule selectively bindsto said internal reference.

Accordingly, the present invention relates to an array comprising asubstrate with predefined regions, wherein each binding substanceimmobilized at a predefined region of said substrate comprises areceptor and a predetermined amount of an internal reference, whereinthe signal generated by said internal reference is determined by meansof a reporter molecule and wherein said reporter molecule selectivelybinds to said internal reference.

Tyras is a method for amplifying RNA by creating, in a non-specificmanner, multiple RNA copies starting from nucleic acid containingstarting material comprising a pool of mRNAs each mRNA comprising apoly-A tail, wherein the material is contacted simultaneously with anoligonucleotide comprising an oligo-dT sequence, the sequence of apromoter recognized by a RNA polymerase and a transcription initiationregion which is located between the oligo-dT sequence and the sequenceof the promoter, and further with an enzyme having reverse transcriptaseactivity, an enzyme having RNase H activity and an enzyme having RNApolymerase activity and the necessary nucleotides and the resultingreaction mixture is maintained under the appropriate conditions for asufficient amount of time for the enzymatic processes to take place.This will lead to the formation of multiple anti-sense RNA copies of themRNAs present in the reaction mixture. Tyras does not involve theproduction of cDNA intermediates; RNA is copied directly from the mRNApresent in the material under investigation. Tyras does not need a cDNAas a basis for the amplification of the RNA. The RNA is synthesized byan RNA polymerase, directly from the mRNA template. The activity of theRNA polymerase is independent from any secondary structures present inthe mRNA and thus there are no differences in the way the differentmRNAs are amplified depending on structures in the mRNAs. The copiesmade represent the original mRNA population as present in the startingmaterial. The oligonucleotides used with Tyras comprise an oligo-dTsequence which will hybridize to the poly-adenylated tail at the 3′ endof the mRNAs. The oligonucleotides further comprise the sequence of apromoter recognized by an RNA polymerase and a transcription initiationregion which is located between the oligo-dT sequence and the sequenceof the promoter. The promoter may be the promoter for any suitable RNApolymerase. Examples of RNA polymerases are polymerases from E. coli andbacteriophages T7, T3 and SP6. In this respect, WO 99/43850 by Pam Geneis exemplary, and is specifically incorporated in the present invention.

The present invention also provides kits for performing the subjectarray-based hybridization assays. The subject kits at least includereporter nucleic acids, as defined above, or a precursor thereof. By“nucleic acid precursor” is meant any nucleic acid from which with thecontrol set may be prepared, e.g. a set of RNAs encoding the nucleicacids of the control set, plasmids containing nucleic acids forgeneration of the control set, and the like.

Labeled cDNA can be derived from these precursors by enzymaticsynthesis, or oligonucleotides chemically synthesized based on sequenceinformation of these precursors. The kits may contain RNAs thatrecognizes each probe composition on an array, and such RNAs may bepre-labeled, may be labeled for use with the analyte nucleic acids, ormay be converted to labeled cDNA for hybridization. Kits of the presentinvention may also contain cDNA or oligonucleotides that selectivelybind to the receptor compositions of the array to be screened. The cDNAsor oligonucleotides may be pre-labeled, or may be labeled by the userthrough any convenient protocol, such as the protocol used to generatethe labeled reporter nucleic acids. A kit containing a set of controltarget RNAs may further contain oligonucleotides for the production ofcDNA. In an exemplary embodiment, these oligonucleotides are genespecific primers, particularly gene specific primers that have sequenceidentical to those that were used in the production of the receptorcompositions on the array to be used in the particular assay. In anotherembodiment, primers can be oligo dT or random primer, if these primersare used for making test sample target.

Kits for carrying out differential gene expression analysis assays arecontemplated. Such kits according to the subject invention will at leastcomprise the subject sets of nucleic acids, e.g. receptors and internalreferences. The kits may further comprise one or more arrayscorresponding to the set of reporter nucleic acids.

The kits may further comprise one or more additional reagents employedin the various methods, such as: primers for generating target nucleicacids; dNTPs and/or rNTPs, which may be either premixed or separate; oneor more uniquely labeled dNTPs and/or rNTPs, such as biotinylated or Cy3or Cy5 tagged dNTPs; or other post synthesis labeling reagents, such aschemically active derivatives of fluorescent dyes, enzymes such asreverse transcriptases, DNA polymerases, RNA polymerases and the like;various buffer mediums, e.g. hybridization and washing buffers;prefabricated probe arrays; labeled probe purification reagents andcomponents, like spin columns, etc.; signal generation and detectionreagents, e.g. streptavidin-alkaline phosphatase conjugate,chemifluorescent or chemiluminescent substrate; and the like.

In addition to the sets of nucleic acids, arrays and other componentsdescribed above in the general description of kits, the assay kit mayfurther include a set of gene specific primers that are employed togenerate labeled analyte nucleic acids. In many embodiments, the set ofgene specific primers will be the same primers used to generate thepolynucleotide receptors that are present on the array to be screened.

Accordingly, the present invention relates to a device or kit comprisinga flow-through based array as described herein.

Accordingly, the present invention relates to the use of a device or kitas described herein, in expression profiling assay, genotyping, sequencedetermination by hybridization, gene quantitation, gene abnormalityanalysis (MAPH), PCR, NASBA, or TYRAS.

Accordingly, the present invention relates to the use of a reportermolecule for the manufacture of or the incorporation into a device orkit as described herein.

Accordingly, the present invention relates to the use of an internalreference for the manufacture of or the incorporation into a kit ordevice as described herein.

Accordingly, the present invention relates to a method for correlatingvariation in analytes, comprising:

-   -   providing at least two analytes, wherein each analyte is        identified according to the method of the present invention,    -   comparing the values of the normalised analytes as defined in        the present invention, whereby variation in analytes is        correlated.

Accordingly, the present invention relates to a method of generating areport that correlates analyte variation determined by a methodaccording to the present invention.

Accordingly, the present invention relates to a computer systemcomprising data obtained according to a method, assay, array or kit ofthe present invention.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described herein, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

The following examples are offered by way of illustration and not by wayof limitation.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Fluorophore for the Reporter Probe

(A) overview of the array; (B) signal detected with an NBB filter g5f20(Narrow band blue filter); (C) signal detected with aWIGfilterg0f1_(—)125 (Super wide band green).

FIG. 2: IRP/Receptor Ratio Optimisation

(A) array overview; (B) sample fluoresceine-signal with a Narrow bandblue filter, after 30 minutes at 770 ms integration time; (C) IRP Texasred signal with a Wide band green filter, after 30 minutes at 440 msintegration time.

FIG. 3: Normalisation of PamChip

(A) array overview; (B) sample signal with Narrow band blue filter,after 30 minutes at 440 ms integration time (inhomogeneous byillumination errors); (3C) IRP signal with Wide band green filer, after30 minutes at 27.5 ms integration time (Inhomogeneous by illuminationerrors); (D) sample signal with Narrow band blue filter, after 30minutes at 770 ms integration time; (E) total illuminated area with anindication of an air bubble on the left part of the image, correspondingto (D); (F) IRP signal with Wide band green filter, after 30 minutes at200 ms integration time; (G) total illuminated area with an indicationof the air bubble on the left part of the image, corresponding to (F).

EXAMPLES Example 1 Materials

Detections were performed utilising fluorescent microscopy (Olympus,Tokyo Japan).

Oligonucleotides were prepared and coupled to the substrate aspreviously described in PCT/EP98/04938. A non-human plant virus sequencefrom the Potato Leafroll RNA Virus (PLRV)-S2 sequence was used asinternal reference IRP; (see Klerks et al. J. Vir. Methods 93(2001)115-125).

Oligonucleotide sequences:

-   -   IRP: PLRV-s2 (SEQ ID NO: 1; tgcaaagtatcatccctccag) (5′        activated)    -   Rho: Reporter probe, 5′-Rhodamine labelled comPLRV_rho (SEQ ID        NO: 2; ctggagggatgatactttgca)    -   Rox: Reporter probe 5′-ROX labelled comPLRV_rox (SEQ ID NO: 3;        ctggagggatgatactttgca)    -   TxR: Reporter probe 5′-Texas Red labelled comPLRV_tex (SEQ ID        NO: 4; ctggagggatgatactttgca);    -   F2: Target sequence 5′-fluorescein labelled F2, (SEQ ID NO: 5;        TCC TTT TCC AGT TCT GTA CAA)    -   R REF1(S2+F), (5′-FAM labelled) designated as R (SEQ ID NO: 6;        catgtatcgaggataaatgaag)    -   HIVpol7p41-3, -5, -6, 10, -16, -18, -20, -22, -23, corresponding        to SEQ ID NOs: 7 9, 10, 11, 12, 13, 14, 16 and 17, respectively        (see Table 2)

Example 2 Fluorophore for the Reporter Probe

In order to simultaneously distinguish reporter binding to the internalreference (IRP) and analyte binding to receptor, respectively, reporterand analyte should be differentially labeled. Below an experiment isgiven with PamGene microarray spots of 300 pL of Rhodamine (Rho), ROX(Rox) and Texas Red (Tx) labelled oligonucleotides (each 10 μM) andFluorescein labelled oligonucleotide (F2) of 1 μM.

The experimental set up was essentially as described in WO 99/02266,which is herein specifically incorporated by reference.

In short, oligonucleotide probes were covalently coupled to the Anoporemembranes using 3-aminopropyl triethoxysilane (APS) as a linker betweenthe alumina and the oligonucleotide.

After rinsing with water, the membranes were dried and immersed in a0.25% (v/v) solution of APS in water for 2 hours. Excess APS was removedby rinsing with water. After drying at 120° C. at reduced pressure themembranes were stored. Amino group concentration due to the coupling ofthe APS molecules was typical 2-3 μmol/m².

Before coupling, the amino group terminated oligo nucleotides wereactivated by reaction with disuccinimidyl suberate (DSS, see eg. PIERCEBV, Immunotechnology Catalog & Handbook, 1990). The resultingsuccinimidyl group at the end of the oligonucleotide was used forcoupling to the APS activated membrane. Coupling with oligonucleotidesolution on an Anopore membrane during 60 minutes resulted in a couplingyield of 1×10⁻¹⁰ mol/m² oligonucleotides.

For detection, fluorescent microscopy was utilised as described inExample 1 (Olympus, Tokyo Japan).

FIG. 1A depicts the overview of the array. FIG. 1B depicts the signalresulting from using an NBB filter g5f20 (Narrow band blue filter). FIG.1C depicts the signal resulting from using a WIGfilterg0f1_(—)125 (Superwide band green).

In order to minimise cross-talk between the fluorophores, thefluorophore used for the reporter probe should preferably have adistinct excitation and emission profile as compared to the fluorophoreused at the target (analyte). As fluorophore of the reporter probepreferentially Texas Red>ROX>Rhodamine should be used in combinationwith a fluorescein sample fluorophore (analyte).

Example 3 IRP/Receptor Ratio Optimisation

The IRP (PLRV-s2; SEQ ID NO: 1) was mixed in different concentrationswith the subject receptor (HIVpol7p41-4; SEQ ID NOs: 8), according toTable 1. The mixtures were subsequently covalently coupled as outlinedin Example 2.

Different ratio's of the IRP and receptor were spotted in three-foldwithin one array, as depicted in FIG. 2A. Next, the microrarray washybridised with a mixture of Tx.R. and the fluoresceine labeled HIVoligo F2, i.e. 20 μl of 1 nM reference probe comPLRV-Texas red (Tx.R.;analyte) and 20 μl of 1 nM reference probe HIV-oligo F2 (reporter) in0.6×SSPE at 45° C. for 30 minutes at 2 pumping steps per minute withsubsequent washing step with 0.6×SSPE at 45° C. The Fluoresceine-signal(by F2) was determined with a Narrow band blue filter (FIG. 2B), whilethe Texas red signal (by Tx.R.) was determined with a Wide band greenfilter (FIG. 2C). TABLE 1 Different ratio's between receptorHIVpol7p41-4 and IRP PLRV-s2. Sample IRP HIVpol7p41-4 PLRV-s2 1 0% 100%2 10% 90% 3 30% 70% 4 50% 50% 5 70% 30% 6 90% 10% 7 100% 0%

Interference from the signal resulting from the IRP-reporter with thesignal resulting from the receptor-reporter is not detectable.Furthermore, interference between the signal resulting from the analytebinding to the receptor and the signal resulting from the IRP binding tothe reporter is not detectable. An amount of 10-90% IRP added to thereceptor may be used.

Example 4 Normalisation of PamChip

An array with 11 different specific receptors, i.e. probes, each havinga different amount of mismatches to the fluorescein labeled target oligo(analyte) was made. The specific receptors are depicted in Table 2.Before spotting, these receptor probes were mixed with the IRP PLRV-s2with a per cent ratio of 70/30. An overview of the array is depicted inFIG. 3A.

Hybridisation as outlined in Example 3, was performed using the sameconditions as outlined above.

Application of the IRP normalisation was performed on deliberatelyinhomogeneous illuminations of arrays. The two methods used were basedon an inhomogeneous light source illumination and inhomogeneous signalsby addition of an air bubble below the array. Image and spot signalintensity determination was done with Array-Pro (MediaCybernetics).TABLE 2 Information of specific receptors (probes) spotted on the arraymismatches SEQ ID No name oligo Sequence with target 7 HIVpol7p4l-3  5′-TTG TAC AGA GAT GGA AAA GGA 2 8 HIVpoI7p4l-4  5′- TTG TAC AGA ACT GGAAAA GGA 0 9 HIVpol7p4l-5  5′- TTG TGC AGA AAT GGA AAA GGA 2 10HIVpol7p4l-6  5′- TTG TAC AGA AAT GGA AAA AGA 2 11 HIVpol7p4l-10 5′- TTGCAC AGA AAT GGA AAA GGA 2 12 HIVpoI7p4l-16 5′- TTG TAC AGA ACT GGA GAAGGA 1 13 HIVpol7p4l-18 5′- TTG TAA AGA GAT GGA ACA GGA 4 14HIVpol7p4l-20 5′- TTG TGC AGA TAT GGA AAA GGA 3 15 HIVpoI7p4l.21 5′- TTGTGC ATT TAT GGA GGA GGA 7 16 HIVpol7p4l-22 5′- TTG TAC AGA ATT GGA AAAGGA 1 17 HIVpol7p4l-23 5′- TTG TTT AGA AAT GGA AAA GGA 3 18 flu labelledtarget 5′- TCC TTT TCC AGT TCT GTA CAANC = negative control, complete different sequenceR = reference, fluorescein labeled oligo for positioning

TABLE 3 Results of the first series of experiments Array SignalSample/IRP CV % Position Oligo Sample IRP Nomalised Sample Normalised1-1:2 6l 1.5 27.9 1.5 47% 43% 1-1:5 6 3.0 30.0 2.8 1-1:3 NCl 0.0 27.10.0 1-1:6 NC 0.1 28.5 0.1 1-2:1 3l 8.2 26.2 8.7 27% 15% 1-2:4 3 11.930.8 10.8 1-2:2 10l 2.3 26.2 2.4 45% 40% 1-2:5 10 4.4 28.4 4.3 1-2:3 22l16.3 27.3 16.6 31% 23% 1-2:6 22 25.3 30.5 23.3 1-3:1 4l 28.5 25.1 31.719%  3% 1-3:4 4 37.5 31.5 33.3 1-3:2 16l 16.1 27.5 16.4 25% 15% 1-3:5 1623.0 31.9 20.2 1-3:3 23l 0.6 24.1 0.6 1-3:6 23 0.4 25.6 0.4 1-4:1 5l13.9 24.0 16.2 12%  8% 1-4:4 5 16.4 31.9 14.4 1-4:2 20l 0.7 27.5 0.7 25%16% 1-4:5 20 1.0 31.3 0.9 1-4:3 18l 1.2 26.6 1.3 1-4:6 18 0.8 25.4 0.928.0 29% 21%

Normalization was done by (Net Sample signal/Net IRP)/Average IRP,wherein the Net Sample signal is the signal resulting from the analyteto the receptor. The effects of normalization were expressed in terms ofaverage variation between duplicate spots.

4.1 The first series of experiments are depicted in FIG. 3B, relating tosample signal with Narrow band blue filter, after 30 minutes at 440 msintegration time (Inhomogeneous by illumination errors), and FIG. 3C,relating to the IRP signal with Wide band green filter, after 30 minutesat 27.5 ms integration time (inhomogeneous by illumination errors). Theresults of the first series of experiments are summarized in Table 3.

Normalisation reduces the variation between spots from 29±12% to 21±14%.Normalisation with the IRP has lead to a 33% lower variation betweenduplicates.

4.2 The second series of experiments relates to environmental influenceson the picture, resulting in bad duplicates. In this case, an air bubblewas created under the slide. The results are depicted in FIGS. 3D-3G.

FIG. 3D depicts the sample signal with Narrow band blue filter, after 30minutes at 770 ms integration time. FIG. 3E, which corresponds to FIG.3D, demonstrates a total illuminated area with an indication of an airbubble on the left part of the image.

FIG. 3F depicts the IRP signal with Wide band green filter, after 30minutes at 200 ms integration time. FIG. 3G, which corresponds to FIG.3F, depicts the total illuminated area with an indication of the airbubble on the left part of the image.

Note, during imaging of the arrays of the first end second series ofexperiments, the air bubble shifter slightly, less than 50 μm, betweenthe two images taken on the array.

The results of the second series of experiments are summarized in Table4.

Normalisation reduces the variation between duplicates from 34±21% to15±6%. Normalization with the IRP has lead to a 50% lower variatonbetween duplicates. TABLE 4 Results of second series of experimentsArray Signal Sample/IRP CV % Position Oligo Sample IRP Nomalised SampleNormalised 1-1:2 6l 16.6 87.6 12.9 48% 17% 1-1:5 6 8.2 34.1 16.3 1-1:3NCl 0.0 68.9 0.0 1-1:6 NC 0.0 36.7 0.0 1-2:1 3l 75.6 123.2 41.7 15% 22%1-2:4 3 61.1 72.8 57.0 1-2:2 10l 30.6 105.6 19.7 49% 26% 1-2:5 10 14.935.8 28.3 1-2:3 22l 98.0 88.5 75.2 54%  6% 1-2:6 22 43.7 36.5 81.5 1-3:14l 144.1 105.1 93.1  3% 13% 1-3:4 4 137.3 83.2 112.2 1-3:2 16l 102.0104.8 66.1 56% 10% 1-3:5 16 45.1 40.1 76.4 1-3:3 23l 0.0 89.7 0.0 1-3:623 1.0 35.2 2.0 1-4:1 5l 87.8 113.7 52.5  5% 11% 1-4:4 5 82.2 90.9 61.41-4:2 20l 5.0 109.1 3.1 45% 14% 1-4:5 20 2.6 46.7 3.8 1-4:3 18l 0.0 81.40.0 1-4:6 18 0.6 38.6 1.0 67.9 34% 15%

1. A method for the identification of an analyte in a biological samplecomprising the steps of: (a) providing a microarray comprising asubstrate with predefined regions wherein each binding substanceimmobilized at a predefined region onto said substrate comprises apredetermined amount of receptor and a predetermined amount of aninternal reference, (b) providing a reporter molecule that bindsselectively to said internal reference, (c) adding said reportermolecule to said biological sample, (d) contacting said biologicalsample comprising said reporter molecule with said microarray underconditions that allow binding to take place between said receptor andsaid analyte, and between said internal reference and said reportermolecule, (e) determining the signal of said reporter molecule bindingto said internal reference, (f) determining the signal of said analytebinding to said receptor, and (g) normalising said signal of step (f)for said signal of step (e), whereby said analyte is identified.
 2. Themethod according to claim 1, wherein said microarray is a flow-throughmicroarray.
 3. The method according to claim 1, wherein said substrateis a porous substrate.
 4. The method according to claim 1, wherein saidsubstrate is an electrochemically manufactured metal oxide membrane. 5.The method according to claim 1, wherein said substrate comprisesaluminium oxide.
 6. The method according to claim 1, wherein saidinternal reference comprises polynucleic acids or (poly)peptides orchemical compounds.
 7. The method according to claim 1, wherein saideach binding substance immobilized onto said substrate comprises atleast 1% to at most 99% of said internal reference.
 8. The methodaccording to claim 1, wherein said each binding substance immobilizedonto said substrate comprises the same predetermined amount of saidinternal reference.
 9. The method according to claim 1, wherein saidreceptor and said internal reference are separate molecules.
 10. Amethod for the normalization of a microarray comprising the steps of:(a) immobilizing onto said array a binding substance comprising areceptor and a predetermined amount of an internal reference, and (b)determining the signal generated by said internal reference by means ofa reporter molecule which selectively binds to said internal reference.11. The method according to claim 10, wherein said reporter moleculecomprises polynucleic acids, (poly)peptides or chemical compounds. 12.The method according to claim 10, wherein said reporter moleculecomprises a label.
 13. The method according to claim 12, wherein saidlabel is of the enzymatic, fluorescent, phosphorescent or radioactivetype.
 14. The method according to claim 10, wherein said internalreference comprises nucleic acids.
 15. The method according to claim 1,wherein said analyte is labeled.
 16. The method according to claim 15,wherein said label is of the enzymatic, fluorescent, phosphorescent orradioactive type.
 17. The method according to claim 1, wherein theanalyte comprises a label, the internal reference comprises a label, andwherein the label of said analyte differs from the label of saidinternal reference.
 18. The method according to claim 17, wherein thelabel of said analyte is Texas red, and the label of said internalreference is fluorescein.
 19. The method according to claim 17, whereinthe label of said internal reference is Texas red, and the label of saidanalyte is fluorescein. 20-23. (canceled)
 24. Microarray comprising asubstrate with predefined regions, wherein each binding substanceimmobilized at a predefined region of said substrate comprises areceptor and a predetermined amount of an internal reference, whereinthe signal generated by said internal reference is determined by meansof a reporter molecule and wherein said reporter molecule selectivelybinds to said internal reference.
 25. Device or kit comprising aflow-through based microarray according to claim
 24. 26-28. (canceled)29. A method for correlating variation in analytes, comprising: (a)providing at least two analytes, wherein each analyte is identifiedaccording to the method according to claim 1, and (b) comparing thevalues of the analytes of step (g) as defined in claim 1, wherebyvariation in analytes is correlated. 30-31. (canceled)
 32. The methodaccording to claim 1, wherein said reporter molecule comprisespolynucleic acids, (poly)peptides or chemical compounds.
 33. The methodaccording to claim 1, wherein said reporter molecule comprises a label.34. The method according to claim 33, wherein said label is of theenzymatic, fluorescent, phosphorescent or radioactive type.